\chapter{Experiment}

\section{03-01: Starting cell culture of 99-24}
\label{sec:03-01}
DATE: 27/10/03

\subsection{Protocol}
Cell culture was initiated according to the protocol \ref{sec:protocol-cell-culture}

\subsection{Results}

The cells were then subject to cell splitting, see Experiment 03-02 (Section \ref{sec:03-02})


\begin{itemize}
\item \textbf{27/10/03} Cell culture began.  
\item \textbf{29/10/03} Cells were examined under the microscope then fed with 10 ml \ac{DMEM}/10\% \ac{CS}.
\item \textbf{31/10/03} Cells were examined under the microscope then fed with 10 ml \ac{DMEM}/10\% \ac{CS}.
\item  \textbf{03/11/03} Cells were examined under the microscope then fed with 10 ml \ac{DMEM}/10\% \ac{CS}.
\item \textbf{05/11/03} Cells were examined under the microscope then fed with 10 ml \ac{DMEM}/10\% \ac{CS}.
\end{itemize}


\section{03-02: Splitting cell culture from 03-01}
\label{sec:03-02}
DATE: 06/11/03

\subsection{Protocol}
The splitting of the cell culture from 03-01 was initiated according to the protocol \ref{sec:protocol-splitting-cell-culture} 

\subsection{Results}

20 plates were produced and the continually fed according to the list below

\begin{itemize}
\item \textbf{7/11/03} Cells were examined under the microscope then fed with 10 ml DMEM/10\% CS 7/11/03.
\item \textbf{10/11/03} Cells were examined under the microscope then fed with 10 ml DMEM/10\% CS 10/11/03.
\item \textbf{12/11/03} Cells were examined under the microscope then fed with 10 ml DMEM/10\% CS 12/11/03.
\item \textbf{14/11/03} Cells were examined under the microscope then fed with 10 ml DMEM/10\% CS 14/11/03.
\end{itemize}

Differentiation of cells occurred 17/11/03  Experiment 03-04, see Section \ref{sec:03-04}.

\section{03-03: Cell culture splitting of one plate from 03-02}
\label{sec:03-03}

DATE: 13/11/03

\subsection{Protocol}
One plate from experiment 03-02 (see Section \ref{sec:03-02}) was selected at random and was subject to splitting acording to the Cell culture splitting protocol \ref{sec:protocol-splitting-cell-culture}

\subsection{Results}
\subsubsection{Feeding}
\begin{itemize}
\item \textbf{14/11/03} Cells were examined under the microscope then fed with 10ml DMEM/10\% CS .
\item \textbf{17/11/03} Cells were examined under the microscope then fed with 10ml DMEM/10\% CS .
\item \textbf{19/11/03} Cells were examined under the microscope then fed with 10ml DMEM/10\% CS .
\end{itemize}

\subsubsection{Progression}
\begin{itemize}
\item \textbf{19/11/03} One plate selected and random was split 19/11/03. Experiment 03-05, see Section \ref{sec:03-05}
\item \textbf{21/11/03} Two plates randomly selected for Snap Freezing 21/11/03. Experiment 03-07, see Section \ref{sec:03-07}.
\item \textbf{21/11/03} Remaining Cells moved to differentiation . Experiment 03-06, see Section \ref{sec:03-06}.
\end{itemize}



\section{03-04: Differentiation of cell culture 03-02 (from Section \ref{sec:03-02}}
\label{sec:03-04}

DATE:17/11/03

\subsection{Protocol}
The culture from 03-02 (Section \ref{sec:03-02}) was differentiated according to the Differentiation protocol (Protocol \ref{sec:differentiation})
\subsection{Results}

\begin{itemize}
\item 17/11/03 Cells were examined under the microscope then fed with 10ml Differentiation medium.
\item 19/11/03 Cells were examined under the microscope then fed with 10ml Insulin Medium.
\item 21/11/03 Cells were examined under the microscope then fed with 10ml Adipocyte Medium.
\item 23/11/03 Cells were examined under the microscope then fed with 10ml Adipocyte Medium.
\item 25/11/03 Cells were examined under the microscope then fed with 10ml Adipocyte Medium.
\item 27/11/03 Cells were examined under the microscope then fed with 10ml Adipocyte Medium.
\item 29/11/03 Cells were examined under the microscope then fed with 10ml Adipocyte Medium.
\item Eight plates were randomly selected and progressed to \ac{TNF} induced insulin resistance. Experiment 03-09 (see Section \ref{sec:03-09})
\item Eight plates were randomly selected and progressed to Insulin Induced insulin resistance. Experiment 03-10 (see Section \ref{sec:03-10})
\item The remaining plate was harvested and snap frozen. Experiment 03-11 (see Section \ref{sec:03-11})
\end{itemize}





\section{03-05: Splitting of cell culture from \ref{sec:03-03}}
\label{sec:03-05}


DATE: 19/11/03

\subsection{Protocol}
The splitting of the cell culture from \ref{sec:03-03} was initiated according to the protocol \ref{sec:protocol-splitting-cell-culture} 

\subsection{Results}

\begin{itemize}
 \item 21/11/03: Cells were examined under the microscope then fed with 10 ml \ac{DMEM}/10\% \ac{CS}.
\item 23/11/03: Cells were examined under the microscope then fed with 10ml DMEM/10\% CS .
\item 25/11/03: Cells were examined under the microscope then fed with 10ml DMEM/10\% CS .
\item Cells reached confluence and were progressed to differentiation Experiment 03-08 (See Section \ref{sec:03-08}).
\end{itemize}




\section{03-06: Differentiation of cell culture from \ref{sec:03-03}}
\label{sec:03-06}

DATE:21/11/03

\subsection{Protocol}
The culture from 03-03 (Section \ref{sec:03-03}) was differentiated according to the Differentiation protocol (Protocol \ref{sec:differentiation}).

\subsection{Results}


\begin{itemize}
 \item 21/11/03: Two Plates of Cells were Snap Frozen at Day 0, before differentiation, and continued on a time course every two Days. Experiment 03-07 (see section \ref{sec:03-07}). Cells were examined under the microscope then fed with 10ml Differentiation medium (see Recipie \ref{sec:diff-medium}).. 

\item 23/11/03: Cells were examined under the microscope. Two plates selected at random and Snap Frozen. Experiment 03-07, Day 2 (see section \ref{sec:03-07}). Remaining cells then fed with 10 ml Insulin Medium (see Recipie \ref{sec:insulin-medium}). 

\item 25/11/03: Cells were examined under the microscope. Two plates selected at random and Snap Frozen. Experiment 03-07, Day 4, (see section \ref{sec:03-07}). Remaining cells then fed with 10ml Adipocyte Medium. (see Recipie \ref{recp:adipopcyte-medium}).  

\item 27/11/03: Cells were examined under the microscope. Two plates selected at random and Snap Frozen. Experiment 03-07, Day 6 (see section \ref{sec:03-07}). Remaining cells then fed with 10ml Adipocyte Medium (see Recipie \ref{recp:adipopcyte-medium}).  

\item 29/11/03: Cells were examined under the microscope. Two plates selected at random and Snap Frozen. Experiment 03-07, Day 8. (see section \ref{sec:03-07}). Remaining cells then fed with 10ml Adipocyte Medium, (see Recipie \ref{recp:adipopcyte-medium}).  

\item 01/12/03: Cells were examined under the microscope. Two plates selected at random and Snap Frozen. Experiment 03-07, Day 10, (see section \ref{sec:03-07}). Remaining cells then fed with 10ml Adipocyte Medium, (see Recipie \ref{recp:adipopcyte-medium}).

\item 03/12/03 Cells were examined under the microscope. The remaining seven plates, selected and Snap Frozen. Experiment 03-07, Day 12, (see section \ref{sec:03-07}).

\end{itemize}

\section{03-07: Snap Freezing of Cell Culture using experiment 03/06 (Section \ref{sec:03-06}}
\label{sec:03-07}

DATE: 21/11/03 

\subsection{Protocol}

The culture from 03-06 (section \ref{sec:03-06} was snap frozen according to the Snap freezing Protocol (see Protocol \ref{sec:snap-freezing}).

\subsection{Results}

Two plates were selected at random an Snap Frozen over a time course every two days.
Day 0 was undifferentiated cells.
Day 2 was the second day of differentiation and so on. From Experiment 03-06.


\begin{table} [ht]
\caption[Results summary: Experiment 03-07]{A sumary of the results of experiment 03-07. }
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Day & Number of aliquots & Date \\
\hline
	0 & 2 & 21/11/03 \\
0 & 2 & 21/11/03 \\
2 & 2 & 23/11/03 \\
4 & 2 & 25/11/03 \\
6 & 2 & 27/11/03 \\
8 & 2 & 29/11/03 \\
10 & 2 & 01/12/03 \\
12 & 7 & 03/12/03 \\
\hline
\end{tabular}
\end{center}
\end{table}



\section{03-08: Differentiation of culture from 03-05}
\label{sec:03-08}

DATE: 27/11/03

\subsection{Protocol}
The culture from 03-05 (Section \ref{sec:03-05}) was differentiated according to the Differentiation protocol (Protocol \ref{sec:differentiation})

\subsection{Results}
\begin{itemize}
 \item 27/11/03 Cells were examined under the microscope then fed with 10 ml Differentiation medium (See Section \ref{sec:diff-medium}).
\item 29/11/03 Cells were examined under the microscope then fed with 10 ml Insulin Medium (See Section \ref{sec:insulin-medium}).
\item 01/12/03 Cells were examined under the microscope then fed with 10 ml Adipocyte Medium (See Section \ref{recp:adipopcyte-medium}).
\item 03/12/03 Cells were examined under the microscope then fed with 10 ml Adipocyte Medium (See Section \ref{recp:adipopcyte-medium}).
\item 05/12/03 Cells were examined under the microscope then fed with 10 ml Adipocyte Medium (See Section \ref{recp:adipopcyte-medium}).

\item Ten Plates were progressed to TNF-alpha Induced Insulin resistance, Experiment 03-13 (See section \ref{sec:03-13}).
\item Ten Plates were progressed to Insulin-Induced Insulin resistance, Experiment 03-14 (See section \ref{sec:03-14}).
\end{itemize}


\section{03-09: TNF-alpha Induced Insulin resistance – of Culture 03-08 (Section \ref{sec:03-08})}
\label{sec:03-09}

DATE:08/12/03

\subsection{Protocol}
From the culture from 03-08 (Section \ref{sec:03-08}), five plates were selected as control and five plates were selected for \ac{TNF} induced insulin resistance, according to the TNF-alpha Induced Insulin resistance protocol (Protocol \ref{sc:tnf-induced}).

\subsection{Results}

\begin{itemize}

\item Visual morphology of cells examined using light microscope.
\item Five Control plates were exposed to the contol protocol (See section \ref{sec:tnf-control-protocol})
\item Five Experiment plates expossed to chronic levels of \ac{TNF} (see section \ref{sec:tnf-exposure-protocol}).
\end{itemize}

\section{03-10: Insulin-induced insulin resistance of Culture 03-04 (Section \ref{sec:03-04})}
\label{sec:03-10}
DATE: 01/12/03

\subsection{Protocol}
From the culture from 03-04 (Section \ref{sec:03-04}), five plates were selected as control and five plates were selected for chronic insulin induced insulin resistance, according to the Insulin Induced Insulin resistance protocol (Protocol \ref{sec:insulin-induced}).

\subsection{Results}


\begin{itemize}
 \item 01/12/03: Visual morphology of cells examined using light microscope. Four Control plates fed with 10ml Adipocyte medium. Four Experiment plates fed with 10ml Adipocyte medium 

\item 03/12/03 Visual morphology of cells examined using light microscope. Four Control plates fed with 10ml Adipocyte medium. Four Experiment plates fed with 10ml Adipocyte medium/10nM Insulin.

\item 04/12/03 Cells harvested after 14 hours and snap frozen. Experiment 03-12 (see Section \ref{sec:03-12})
\end{itemize}


\section{03-11: Snap Freezing of Cell Culture using experiment 03-04 (Section \ref{sec:03-04})}
\label{sec:03-11}

DATE: 01/12/03 

\subsection{Protocol}

The culture from 03-04  was snap frozen according to the Snap freezing Protocol (see Protocol \ref{sec:snap-freezing}).

\subsection{Results}

One plate from culture 03-04,(section \ref{sec:03-04}) not progressed to insulin resistance was selected and snap frozen.

Day 14 of Differentiation	01/12/03


\section{03-12: Snap Freezing of Cell Culture using 03-10 (See Section \ref{sec:03-10})}
\label{sec:03-12}

DATE: 04/12/03 

\subsection{Protocol}

The culture from 03-10  was snap frozen according to the Snap freezing Protocol (see Protocol \ref{sec:snap-freezing}).

\subsection{Results}

Four control plates were snap Frozen and Four Insulin resistant plates were frozen from culture 03-10,(section \ref{sec:03-10}).






\section{03-13: Snap Freezing of Cell Culture using 03-09 (See Section \ref{sec:03-09}}
\label{sec:03-13}

DATE: 04/12/03 

\subsection{Protocol}

The culture from 03-09  was snap frozen according to the Snap freezing Protocol (see Protocol \ref{sec:snap-freezing}).

\subsection{Results}

Four control plates were snap Frozen and Four Insulin resistant plates were frozen from culture 03-09,(section \ref{sec:03-09}).




\section{03-14: TNF-alpha Induced Insulin resistance of Culture 03-08 (See Section \ref{sec:03-08})}
\label{sec:03-14}

DATE: 08/12/03

\subsection{Protocol}
This is the continous feeding of experiment 03-09 (See Section \ref{sec:03-09})

\subsection{Results}

DATE: 08/12/03

\begin{itemize}
 \item Visual morphology of cells examined using light microscope.
\item Five Control plates fed with 10ml Adipocyte medium (See Recipie \ref{recp:adipo-insulin}).
\item Five Experiment plates fed with 10ml Adipocyte medium and 30ng TNF-alpha (See Section \ref{recp:tnf-adipo-medium}).
\end{itemize}


DATE: 09/12/03
\begin{itemize}
 \item Visual morphology of cells examined using light microscope.
\item Five Control plates fed with 10ml Adipocyte medium (See Recipie \ref{recp:adipopcyte-medium}).
\item Five Experiment plates fed with 10ml Adipocyte medium and 30ng TNF-alpha (See Section \ref{recp:tnf-adipo-medium}).
\end{itemize}

DATE: 10/12/03
\begin{itemize}
 \item Visual morphology of cells examined using light microscope.
\item Five Control plates fed with 10ml Adipocyte medium (See Recipie \ref{recp:adipopcyte-medium}).
\item Five Experiment plates fed with 10ml Adipocyte medium and 30ng TNF-alpha (See Section \ref{recp:tnf-adipo-medium}).
\end{itemize}

	
DATE: 11/12/03


\begin{itemize}
 \item Visual morphology of cells examined using light microscope.
\item One Control plate was harvested and Snap Frozen. Experiment 03-17 (See Section \ref{sec:03-17}).
\item One Experiment plate was harvested and Snap Frozen. Experiment 03-17 (See Section \ref{sec:03-17}).
\end{itemize}


The remaining plates: Four Control and Four Experimental plates were progressed to Subcellular Fractionation. Experiment 03-16 (See Section \ref{sec:03-16}).


\section{03-15:Insulin-induced insulin resistance of Culture 03-08 (See Section \ref{sec:03-08})}
\label{sec:03-15}

DATE: 08/12/03

\subsection{Protocol}
Continuation of experiment 03-10 (See Section \ref{sec:03-10}). Five plates were selected as control and five plates were selected for chronic insulin induced insulin resistance, according to the Insulin Induced Insulin resistance protocol (Protocol \ref{sec:insulin-induced}).

\subsection{Results}

DATE: 08/12/03

\begin{itemize}
 \item Visual morphology of cells examined using light microscope.
\item Five Control plates fed with 10ml Adipocyte medium  (See Recipie \ref{recp:adipopcyte-medium}).
\item Five Experiment plates fed with 10ml Adipocyte medium (See Recipie \ref{recp:adipopcyte-medium}).
\end{itemize}

DATE: 10/12/03


\begin{itemize}
 \item Visual morphology of cells examined using light microscope.
 \item Five Control plates fed with 10ml Adipocyte medium (See Recipie \ref{recp:adipopcyte-medium}).
\item Five Experiment plates fed with 10ml Adipocyte medium, 10nm Insulin (See Recipie \ref{recp:adipo-insulin}).
\end{itemize}

After 14 hours:

DATE: 11/12/03

\begin{itemize}
 \item Visual morphology of cells examined using light microscope.
\item One Control plate was harvested and Snap Frozen. Experiment 03-16 (See Section \ref{sec:03-16}).
\item One Experiment plate was harvested and Snap Frozen. Experiment 03-16 (See Section \ref{sec:03-16}).
\end{itemize}

This left four Control Plates and four Experimental Plates, which were progressed to  Sub-cellular Fractionation. Experiment 03-17. (See Section \ref{sec:03-17}).


\section{03-16:Subcellular fractionation of 3T3-L1 adipocytes from 03-14 (Section \ref{sec:03-14}) and 03-15 \ref{sec:03-15})}
\label{sec:03-16}

\subsection{Protocol}
The Subcellular Fractionation protocol (See Protocol \ref{sec:intracell-fraction}) was applied to the cultures from 03-14 (Section \ref{sec:03-14}) to produce the following fractions.


\subsection{Results}


\begin{enumerate}
 \item Control Ins/Ins
 \item Ins/Ins
 \item Control TNF/Ins
  \item TNF/Ins
  \item Control Ins/Basal
  \item Ins/Basal
  \item Control TNF/Basal
  \item TNF/Basal
\end{enumerate}
Each Number corresponds to the following conditions


\textbf{Control} : is a control plate\\
\textbf{Ins/Ins}	:Insulin-Induced insulin resistance, stimulated for 20 minutes with 
10nM Insulin prior to harvesting.\\
\textbf{TNF/Ins}:TNT-alpha-induced insulin resistance, stimulated for 20 minutes with 
10nM Insulin prior to harvesting.\\
\textbf{Ins/Basal}:I-I insulin resistance, without excess insulin prior to harvesting	\\
\textbf{TNF/Basal}:TNF-insulin resistance, without excess insulin prior to harvesting\\

\subsubsection{\ac{HDM}}

The \ac{HDM} pelletes were collected and resuspended in 500$\mu$l of \ac{HES}. They were then coded in the following way indicating which plate they originated from and frozen.  \texttt{Experiment.HDM.Plate}

\begin{itemize}
 \item 03-16.HDM.1
 \item 03-16.HDM.2
 \item 03-16.HDM.3
 \item 03-16.HDM.4
 \item 03-16.HDM.5
 \item 03-16.HDM.6
 \item 03-16.HDM.7
 \item 03-16.HDM.8

\end{itemize}

\subsubsection{LDM}

The \ac{LDM} pellets were resuspended in 500$\mu$l of \ac{HES}. They were then coded in the following way indicating which plate they originated from and frozen.


\begin{itemize}
 \item 03-16.LDM.1
\item 03-16.LDM.2
\item 03-16.LDM.3
\item03-16.LDM.4
\item 03-16.LDM.5
\item 03-16.LDM.6
\item 03-16.LDM.7
\item 03-16.LDM.8

\end{itemize}

\subsubsection{Cytosol}

A 2ml of the cytolsol supernatant were collected. They were then coded in the following way indicating which plate they originated from and frozen.

\begin{itemize}
 \item 03.16.CY.1
 \item 03.16.CY.2
 \item 03.16.CY.3
 \item 03.16.CY.4
 \item 03.16.CY.5
 \item 03.16.CY.6
 \item 03.16.CY.7
 \item 03.16.CY.8
\end{itemize}

\subsubsection{\ac{M/N}}

The M/N pellets were resuspended in 500$\mu$l of \ac{HES}. They were then coded in the following way indicating which plate they originated from and frozen.


\begin{itemize}
 \item 03.16.MN.1
 \item 03.16.MN.2
 \item 03.16.MN.3
 \item 03.16.MN.4
 \item 03.16.MN.5
 \item 03.16.MN.6
 \item 03.16.MN.7
 \item 03.16.MN.8

\end{itemize}

\subsubsection{\ac{PM}}

The \ac{PM} pellets were resuspended in 500$\mu$l of \ac{HES}. They were then coded in the following way indicating which plate they originated from and frozen.


\begin{itemize}
 \item 03.16.PM.1
 \item 03.16.PM.2
 \item 03.16.PM.3
 \item 03.16.PM.4
 \item 03.16.PM.5
 \item 03.16.PM.6
 \item 03.16.PM.7
 \item 03.16.PM.8
\end{itemize}



\section{03-17: Snap Freezing of Cell Culture using 03/15 (Section \ref{sec:03-15}) and 03-14 (Section \ref{sec:03-14})}
\label{sec:03-17}


DATE: 11/12/03 

\subsection{Protocol}

Two plates from Experiment 03-15 (Section \ref{sec:03-15}) (one control and one Experiment plate) and Two plates from Experiment 03-14,Section \ref{sec:03-14}) (one control and one Experiment plate) were snap frozen according to the Snap freezing Protocol (see Protocol \ref{sec:snap-freezing}).

\subsection{Results}


A total of four plates were harvested and snap frozen, coded and stored in the freezer

\begin{table} [ht]
\caption[Results summary: Experiment 03-17]{A sumary of the snap freezing aliquots of experiment 03-17. }
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Experiment & Plate & Aliquot Identifier \\
\hline
	03-14 & TNF-alpha Control & 03-17.a \\
03-14 & TNF-alpha  & 03-17.b \\
	03-15 & Insulin Control & 03-17.c \\
03-15 & Insulin & 03-17.d \\

\hline
\end{tabular}
\end{center}
\end{table}

\section{03-18: Lowry Procedure Using Samples from 03-07}

\subsection{Protocol}

Seven aliquots from Day 12 of Experiment 03-07, (Section \ref{sec:03-07}) were defrosted and pooled together. 500 $\mu$l were used in this protein assay, (See Protocol \ref{sec:lowry}) the rest were frozen down in 1 ml aliquots.

\subsection{Results}

The protein standards were made up as in Table \ref{tab:protein-stand}. The volume of buffer was added so to match the concentration of the cells in the sample. Each experiment was repeated twice.


\begin{table} [ht]
\caption[Lowry Protein concentration]{Lowry protein concentration}
\begin{center}

\begin{tabular}{| p{3cm} |p{3cm} |p{3cm} |p{3cm} | p{3cm} }
\hline
  Tube & Volume of stock ($\mu$l) & H20 ($\mu$l) & Buffer ($\mu$l) & Protein Concentration ($\mu$g/ml)\\
\hline
	1 & 0 & 850 & 150 & 0 \\
	2 & 25 & 825 & 150 & 10\\
	3 & 50 & 800 & 150 & 20 \\
	4 & 100 & 750 & 150 & 40 \\
	5 & 150 & 700 & 150 & 60 \\
	6 & 200 & 650 & 150 & 80 \\
	7 & 250 & 600 & 150 & 100 \\
	8 & 375 & 475 & 150 & 150 \\
	9 & 500 & 350 & 150 & 200\\
\hline
\end{tabular}
\end{center}
\label{tab:protein-stand}
\end{table}


Samples were made up as in Table \ref{tab:protein-stand-sample} and the experiment was repeated 3 times.
Assuming the concentration of protein in the cell sample is 6$\mu$g/ml therefore


\begin{itemize}
 \item 10     $\mu$g/ml = 1.5  $\mu$l
\item 100  $\mu$g/ml = 15  $\mu$l
\item 1000 $\mu$g/ml = 150 $\mu$l
\end{itemize}

\begin{table} [ht]
\caption[Lowry Protein concentration for samples]{Lowry protein concentration for samples}
\begin{center}

\begin{tabular}{| p{3cm} |p{3cm} |p{3cm} |p{3cm} | p{3cm} }
\hline
  Tube & Sample ($\mu$l)  & Buffer ($\mu$l) & H20 ($\mu$l) \\
\hline
	A & 1.5 & 148.5 & 850  \\
	B & 15 & 135 & 850 \\
	C & 150 & 0 & 850 \\
	
\hline
\end{tabular}
\end{center}
\label{tab:protein-stand-sample}
\end{table}


